The family of Toll like receptors (TLRs) is involved in the detection of most classes of pathogens. TLRs on the cell surface recognize conserved molecules of bacteria fungi and parasites that are “foreign” to the host organism [1]. A structurally closely related subset of TLRs has been shown to contribute to the detection of viruses [2-5]. Viruses derive all their components from the infected host cell and therefore do not contain “foreign” molecules. Detection of viruses instead hinges on the strategic localization of TLRs that recognize viral nucleic acids in endosomes and lysosomes. These cellular compartments are not usually accessed by host nucleic acids, but are traversed by most viruses during their infectious cycles. The endosomal TLRs 3, 7/8 and 9 have been shown to recognize double stranded (ds), single stranded (ss) or short double stranded RNA and DNA, respectively [2,6-8]. In humans, the function to recognize viral ssRNA is shared by TLR7 and TLR8 [7], and the expression pattern of both receptors appears mutually exclusive, and is largely confined to immune cell subsets. Among human PBMC, TLR7 is expressed by plasmacytoid dendritic cells (PDCs) and by B cells, while monocytes and myeloid dendritic cells express TLR8 [9,10]. In contrast, mice deficient in TLR7 were found entirely unable to respond to ssRNA ligands, indicating that mouse TLR8 is either inactive, or has a non-immune function [11,12]. The first defined TLR7/8 ligands were imidazoquinoline derivates and C8 and/or N7 modified analogs of guanosine, small molecules known to induce an antiviral response [11,13-15]. Most of these compounds (e.g. resiquimod R-848, 3M-003) activate both, TLR7 and TLR8. Others, including imiquimod R-837, 3M-001 and loxoribine, are TLR7 selective, and mainly stimulate PDC to produce IFN-α, or preferentially activate TLR8 (3M-002), and induce monocytes to secrete TNF-α and IL-12. The differential cytokine profiles are of relevance for the therapeutic use of these compounds. The first drug of this class is 5% imiquimod cream, which is approved for the topical treatment of genital warts caused by human papillomavirus (HPV), as well as of basal cell carcinoma and actinic keratosis [16]. Recognition of cognate RNA ligands by TLR7/8 has been studied mostly using short synthetic oligoribonucleotides (ORNs) [17-22]. To achieve stimulation, ORN must be formulated in a complex with lipid transfection reagents or polycations to enable delivery to the endosomal compartment [6,7,21,23]. It was noted that ORN lacking uridines were not immunostimulatory, and most ORNs rich in guanosines and uridines were stimulatory for both, TLR7 and TLR8 [7]. Stimulatory sequence motifs were reported [21], however a systematic evaluation of sequence motifs has not been performed. Based on the observation that homopolymeric uridine (pU) and ORNs consisting of 21 uridine repeats show TLR7 ligand activity, it was proposed that bona fide RNA recognition motifs may not exist [18]. However, a recent publication confirmed that some ORN sequences lacking guanosines selectively activate TLR8 [20]. Other studies found that compared to the single stranded ORNs, complementary strands in the siRNA duplex show reduced TLR8 activity [24]. The biological impact of a differential recognition of RNA by TLR7 and TLR8 is unknown, and selective activation of human TLR7 by RNA has so far not been reported. Natural ssRNAs including mRNA and viral RNA do not present as a single strand under physiological conditions but form secondary structures in which the majority of bases are paired in double helical stems to minimize free energy [25]. Besides the standard Watson-Crick base pairing RNA secondary structure additionally contains wobble (i.e. non-canonical) base pairs formed by guanosines and uridines [26]. G·U (guanosine.uridine) base pairs can functionally substitute for Watson-Crick base pairs, as they are nearly isomorphic, and of comparable thermodynamic stability [27]. RNA stem structures containing G·U wobble base pairs are present in virtually all functional RNA classes and are therefore a hallmark of ssRNA secondary structure. Moreover, in a wide range of biological processes, unique structural, chemical and conformational characteristics mark sites containing G·U base pairs for recognition by proteins [27,28].
Here, we examined whether RNA secondary structural elements, in particular those that form G·U wobble base pairs, influence ssRNA recognition. We found that short RNA stems were highly immunostimulatory, and selectively activated TLR7 when they contained at least one G·U wobble base pair. The G·U base pair therefore constitutes the so far unappreciated minimal structural motif sufficient to confer TLR7 agonist activity in single stranded RNA. In addition, we identified the first phosphodiester RNA ligand, which selectively stimulates TLR7 on its own, obviating the need for formulation with transfection reagents such as polycations.
Therefore, there is a need in the art to better understand the mechanism by which TLR7 distinguishes between self and non-self RNA. Specifically, there is a need in the art to provide molecules that are specifically recognized by TLR7 and/or activate TLR7. The provision of such molecules allows for the provision of immunostimulatory nucleic acid molecules, which are useful for the production of type I IFN in vitro and in vivo and for treating various diseases, which can be alleviated or even eradicated by type I IFN, such as viral infections, immune disorders and cancers.